Fusarium oxysporim M1菌株的rDNA ITS鉴定及其纤维素酶活性及纯化研究

以1株分离于北大仓白酒大曲的产纤维素酶真菌M1为材料,对其进行了形态及分子生物学鉴定;纯化并研究了其纤维素酶的酶学性质。以真菌ITS1/ITS4通用引物,扩增真菌M1的rDNA ITS序列,再与GenBank中其他菌株rDNA ITS序列进行比对,使用Mega5.0软件,采用最大似然法进行聚类分析。结果显示,该真菌同已经报道的Fusarium oxysporim strain Bt3聚为一类,一致率达99%,与形态学方法鉴定一致,命名为Fusarium oxysporum M1。该菌具有很高纤维素酶活力,FPA和CMCA分别高达16.84 IU/mL和35.31 IU/mL。经过发酵条件优化酶活性进一步提高。经硫酸铵分级分离、疏水和离子交换层析,纯化了该菌纤维素酶,纯化倍数高达17.97倍,得率为3.676%,SDS-PAGE分析表明,该纤维素酶分子量达60 k Da。本研究为进一步研究该酶高效催化机理及实际应用提供参考。     

Enhancing cellulase production by overexpression of xylanase regulator protein gene,xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.     

东亚飞蝗肠道内高产纤维素酶菌株筛选及鉴定

为微生物制剂生产筛选菌种资源。运用昆虫解剖技术取出东亚飞蝗(Locusta migratoria manilensis)肠道,采用稀释涂板法对肠道内的菌种进行分离,并利用羧甲基纤维素钠(CMC-Na)改良培养基初筛产纤维素酶菌株。结果表明,从东亚飞蝗肠道内共分离得到12株产纤维素酶菌株,均为细菌,并对纤维素酶活较高的菌株K005进行了形态学和分子生物学鉴定,通过菌落及菌体形态特征、生理生化特性、16S r DNA序列测定结果,将该菌株鉴定为蜡样芽胞杆菌(Bacillus cereus)。     

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L^?1 for rice straw, wheat bran, peptone, and CaCO₃, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL^?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Large-Scale Analyses of Glycosylation in Cellulases

Cellulases are important glycosyl hydrolases (GHs) that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β (1→4) bonds,and they are widely used to produce cellulosic ethanol from the plant biomass.N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency,cellulose binding affinity and the stability of cellulases based on observations of individual cellulases.As far as we know,there has not been any systematic analysis of the distributions of N-...     

Efficient production of cellulase in the culture of Acremonium cellulolyticus using untreated waste paper sludge

Cellulase was produced by Acremonium cellulolyticus using untreated waste paper sludge (PS) as the carbon source. The clay present in PS did not show any inhibitory effect on cellulase production but did alter the pH during fermentation. On the flask scale, the maleate buffer concentration and pH were key factors that affected the efficiency of cellulase production from PS cellulose. Optimum cellulase production in a 3-L fermentor of working volume 1.5 L was achieved by controlling the pH value at 6.0 using 2 M NaOH and 2 M maleic acid, and the productivity reached 8.18 FPU/mL. When 40.89 g/L PS cellulose, 2.2 g/L (NH(4) )(2) SO(4) , and 4.4 g/L urea were added to a 48-h culture, the cellulase activity was 9.31 FPU/mL at the flask scale and 10.96 FPU/mL in the 3-L fermentor. These values are ～80% of those obtained when pure cellulose is used as the carbon source. The method developed here presents a new route for the utilization of PS.